Thrombomodulin analogs for use in recovery of spinal cord injury

ABSTRACT

The present invention relates to the use of thrombomodulin analogs for the treatment of trauma-induced spinal cord injury.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/229,714, filed Aug. 31, 2000, which is incorporatedherein in full by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to a method of using analogs ofthrombomodulin in the treatment of the neurologic trauma associated withspinal cord injury in mammals.

BACKGROUND OF THE INVENTION

[0003] Thrombomodulin (TM) is a cell membrane glycoprotein. In humans,it is widely distributed on the endothelium of the vasculature andlymphatics. Its physiological importance has been extensively studied.(See, for example, Esmon et al., J. Biol. Chem. (1982), 257:859-864;Salem et al., J. Biol. Chem. (1983), 259:12246-12251).

[0004] Thrombomodulin functions as a receptor for thrombin, a centralenzyme in the coagulation cascade. When free, thrombin promotescoagulation both directly by converting fibrinogen to fibrin, indirectlythrough activation of other proteins in the coagulation cascade (FactorsV, VIII and XIII, for example), and through platelet activation. Whenbound to thrombomodulin, however, the thrombin-thrombomodulin complex isinvolved in activation of protein C to activated protein C, which thendownregulates the coagulation cascade by proteolytically inactivatingthe essential cofactors Factor Va and Factor VIIIa (Esmon et al., Ann.N.Y. Acad. Sci. (1991), 614:30-43), resulting in increased anticoagulantactivity. The thrombin-thrombomodulin complex also is involved inactivation of thrombin-activatable fibrinolysis inhibitor (TAFI), which,when activated, leads to inhibition of fibrinolysis. Although earlierstudies were negative, more recent studies have indicated thatthrombomodulin is not only present in brain endothelial cells (Boffa, etal., Nouv. Rev. Fr. Hematol. (1991), 33:423-9; Wong, et al., Brain Res.(1991), 556:1-5; Wang, et al., Arterioscler. Thromb. Vasc. Biol. (1997),17: 3139-46; Tran, et al., Stroke (1996), 27:2304-10; discussion 2310-1)but also is expressed on the surface of astrocytes, where it functionsidentically to its role in the vasculature, activating protein C byforming a complex with thrombin (Pindon, et al., Glia (1997),19:259-68). Thrombomodulin is also upregulated in reactive astrocytes inthe CNS, in response to mechanical injury (Pindon, et al., J. Neurosci.(2000), 20:2543-50). A recent report suggests that recombinantthrombomodulin block thrombin's activation of another receptor, theprotease-activated receptor 1 (PAR-1) in cultured neuronal cells(Sarker, et al. Thromb. Haemost. (1999), 82: 1071-77).

[0005] Activated protein C has also been strongly implicated in theregulation of inflammatory responses involving various cytokines oractivated leukocytes ( Esmon et al., Thromb. Haemost. (1991),66:160-165). Consistent with this hypothesis, studies have shown thatactivated protein C prevents pulmonary vascular injury in rats givenendotoxin by inhibiting production of tumor necrosis factor (TNF-α),which potently activates neutrophils (Murakami et al., Blood (1996),87:642-647; Murakami et al., Am. J. Physiol. (1996), 272:L197-2).Recombinant human soluble thrombomodulin also prevents endotoxin-inducedpulmonary vascular injury by inhibiting the activation of neutrophilsthrough protein C activation (Uchiba et al., Am. J. Physiol. (1996),271:L470-5; Uchiba et al., Am. J. Physiol. (1997), 273:L889-94).

[0006] Spinal cord injury (SCI) is a serious condition which produceslife-long disabilities (Stover et al., Paraplegia (1987), 24:225-228).Only limited therapeutic measures are currently available for itstreatment (Bracken et al., New Engl. J. Med. (1990), 322:1405-1411). Infact, the most commonly accepted acute intervention after SCI, otherthan surgery, is administration of the steroid, methylprednisolone (MP)(Hall, E. D., Adv. Neurol. (1993), 59: 241-8; Bracken, M. B., J.Neurosurg. (2000), 93:175-9; Bracken, M. B., Cochrane Database Syst.Rev. 2 (2000); Koszdin, et al., Anesthesiology (2000), 92:156-63).However, after 10 years of experience this treatment is still quitecontroversial and a recent meta analysis has suggested that treatmentwith MP may actually be contraindicated (Hurlbert, R. J., J. Neurosurg.(2000), 93:1-7; Pointillart, et al., Spinal Cord (2000), 38:71-6;Lankhorst, et al., Brain Res. (2000), 859:334-40).

[0007] The pathophysiology of SCI includes a primary mechanical injuryand a delayed secondary neurological injury (Tator et al., J. Neurosurg.(1991), 75:15-26). Whereas the primary injury is determined by thecircumstances of the trauma, the outcome of the secondary injury may beamenable to therapeutic modulation. Although the mechanisms involved inthe secondary injury process are not fully understood, inflammatoryresponses leading to endothelial damage may be involved (Demopoulos, etal., Scan. Electron Microsc. (1978), 2:677-680) and this is an areawhich can serve as a target for therapeutic intervention. Tumor necrosisfactor (TNF-α) has recently been shown to play an important role incompression trauma-induced SCI in rats by activating neutrophils (Taoka,et al., Neuroscience (1997), 79:1177-182; Taoka et al., J Neurotrauma(2000), 17:219-29). It has also been reported that activated protein Creduces the severity of compression trauma-induced SCI by inhibitingTNF-α production (Taoka et al., J. Neurosci. (1998), 18:1392-1398).Studies have shown that recombinant soluble thrombomodulin preventedcompression trauma-induced SCI in a rat SCI model by inhibitingleukocyte accumulation through reduction of TNF-α mRNA expression at theinjured site (Taoka et al., Thromb. Haemost. (2000), 83:462-468). Theseobservations suggest that thrombomodulin may also prevent contusiontrauma-induced SCI through activation of protein C, with the resultantinhibition of TNF-α production. Contusion, or weight drop rat modelshave recently been validated for human SCI (Metz et al., J Neurotrauma(2000), 17: 1-17).

[0008] We have discovered that certain soluble thrombomodulincompositions are effective in reducing the neurologic damage followingSCI in a rat model, and are therefore useful in the treatment of suchneurologic damage in mammals. Soluble analogs of thrombomodulin thatretain most, if not all, of the activities of the native protein havebeen produced. Furthermore, soluble analogs of thrombomodulin which areresistant to oxidation, resistant to proteolysis, or have in other waysbeen modified so as to possess a longer half-life within thecirculation, have been developed and are described in U.S. Pat. Nos.5,256,770, 5,863,760 and 5,466,668, which are incorporated herein byreference. These compositions have previously been described as beinguseful as anti-thrombotic agents. However, there has not been anydisclosure as to the usefulness of these compositions as therapeuticagents for the amelioration of neurologic damage following SCI.

SUMMARY OF THE INVENTION

[0009] In accordance with the present invention, one aspect of thisinvention is directed to a method for treating the neurologic damageresulting from SCI which method comprises administering to a mammal,most preferably a human, in need thereof, a therapeutically effectiveamount of a soluble, recombinant thrombomodulin analog which isresistant to oxidation and wherein the methionine at position 388 hasbeen replaced with a leucine, wherein the analog is numbered inaccordance with native thrombomodulin (SEQ ID NO:2)

[0010] A further aspect of this invention utilizes thrombomodulinanalogs which contain additional modifications to provide resistance toprotease cleavage and/or show an altered pattern of glycosylation.

[0011] A further aspect of this invention utilizes a thrombomodulinanalog, known as Solulin™, which contains modifications to the sequenceof native thrombomodulin (SEQ ID NO: 2) at the following positions:removal of amino acids 1-3, M388L, R456G, H457Q, S474A, and terminationat P490.

[0012] A further aspect of this invention is directed to apharmaceutical composition useful in treating neurologic damageresulting from spinal cord injury in a mammal, which pharmaceuticalcomposition comprises a pharmaceutical excipient and a therapeuticallyeffective amount of a soluble, recombinant thrombomodulin analog whichis resistant to oxidation and wherein the methionine at position 388 hasbeen replaced with a leucine, wherein the analog is numbered inaccordance with native thrombomodulin (SEQ ID NO: 2).

[0013] Further aspects of the invention are directed to pharmaceuticalcompositions comprising other thrombomodulin analogs which have beenmodified as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 shows the amino acid sequence of native thrombomodulin (SEQID NO: 2), using the numbering system of Suzuki et al. (1987) Embo J 6:1891-1897.

[0015]FIG. 2 shows results of evaluation of neural function by theopen-field locomotor rating scale (LRS) of Basso, Beatty and Bresnahan,the so-called “BBB” scale, in rats following controlled contusion spinalcord injury. Injured rats are either treated with vehicle (saline) orwith Solulin™ administered intraperitoneally (i.p.) post-injury asdescribed in Example 2.

[0016]FIG. 3 (A and B) illustrates representative histologic specimensfrom rats with spinal cord injury following treatment with saline(Control) or with Solulin™ given i.p at 1 h following moderate contusionSCI (25 gm.cm force). Specimens from above, at, and below the injury areshown. In FIG. 3A, the specimens were stained with hematoxylin and eosin(H&E); in FIG. 3B, the specimens were stained with thionin.

[0017]FIG. 4 shows analyses of the extent of lesion volume as determinedby histological examination in saline (control) versus Solulin™ treatedrats, at, above and below the lesion epicenter. Astatistically-significant reduction of about 40% (p<0.05) in lesionvolume was found with Solulin™ treatment as compared to saline treatedcontrols with an unpaired t-test.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Definitions

[0019] As used in the specification and claims, unless specified to thecontrary, the following terms have the meaning indicated:

[0020] The term “residue” refers to an amino acid that is incorporatedinto a peptide. The amino acid may be a naturally occurring amino acidand, unless otherwise limited, may encompass known analogs of naturalamino acids that can function in a similar manner as naturally occurringamino acids. For purposes of this disclosure, amino acid residues aredesignated herein by their accepted three-letter or one-letterabbreviation, or by the notation “AA”, which signifies the presence ofan amino acid residue. The amino acids referred to herein are describedby shorthand designations as follows: TABLE 1 Amino Acid NomenclatureName 3-letter 1 letter Alanine Ala A Arginine Arg R Asparagine Asn NAspartic Acid Asp D Cysteine Cys C Glutamic Acid Glu E Glutamine Gln QGlycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine LysK Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser SThreonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

[0021] When describing an amino acid substitution, for purposes of thisdisclosure, the substitution is described by providing the amino acidpresent in native thrombomodulin (SEQ ID NO: 2) (TM), the location ofthe amino acid within the thrombomodulin sequence (using the numberingsystem of Suzuki et al, Embo Journal (1987), 6:1891-1897), followed bythe amino acid which has been substituted for the original: i.e. M388Lrefers to substitution of methionine at position 388 with leucine).

[0022] “Native thrombomodulin” refers to the full length protein as itoccurs in nature (FIG. 5: SEQ ID NO: 2). Native thrombomodulin is knownto contain naturally occurring polymorphisms at certain residues. Forexample, at position 455, there is a naturally occurring variation inthe amino acid found at this position, with an alanine present 82% ofthe time and a valine present 18% of the time (Van der Velden et al.(1991) Throm. Haemeostasis 65:511-513.) For purposes of this invention,the native thrombomodulin sequence shown (FIG. 5; SEQ ID NO: 2) is onewhich contains valine at position 455, as described by Suzuki et al.(1987) EMBO J 6:1891-1897. However, all naturally occurringpolymorphisms are included within the scope of the claimed analogs. Whenbiological activities are described with reference to the native TM, theterm embraces a detergent solubilized aqueous form. Often, in thecontext of comparison to an activity, a transfected soluble polypeptidemay exhibit substantially identical properties.

[0023] “Thrombomodulin analogs” are peptides which substantially retainthe biological activity of native TM, as discussed above, and which havea molecular structure different from that of a native version TM. Forexample, the term refers to proteins having an amino acid sequenceidentical or homologous with that of native thrombomodulin (SEQ ID NO:2), to insoluble and soluble thrombomodulin peptides or fragments, andto oxidation resistant TM species, all having thrombomodulin-likeactivity. These compounds also include derivatives and moleculescomprising amino acid changes which do not significantly decrease theprotein C activation cofactor properties of the protein when comparedwith native TM.

[0024] The term “TM mutant” refers to a TM analog containing thedesignated substitution (as described above) or other indicatedmodification.

[0025] The terms “peptides” and “polypeptides” refer to chains of aminoacids whose a carbons are linked through peptide bonds formed by acondensation reaction between the α carbon carboxyl group of one aminoacid and the amino group of another amino acid. The terminal amino acidat one end of the chain (amino terminus) therefore has a free aminogroup, while the terminal amino acid at the other end of the chain(carboxy terminus) has a free carboxyl group.

[0026] The term “domain” refers to a discrete amino acid sequence thatcan be associated with a particular function or characteristic.Typically, a domain exhibits a characteristic tertiary structural unit.The full-length thrombomodulin gene encodes a precursor peptidecontaining the following domains: TABLE 2 TM Domains Approximate AminoAcid Position Domain (−18)-(−1)  Signal sequence  1-226 N-terminaldomain (lectin domain; L) 227-462 6 EGF-like domains (E) 463-497O-linked Glycosylation (D) 498-521 Transmembrane 522-557 Cytoplasmicdomain

[0027] See Yost et al., Cell, (1983), 34:759-766 and Wen et al.,Biochemistry (1987), 26:4350-4357, both incorporated herein byreference.

[0028] A “protease site” refers to an amino acid or series of aminoacids in a TM polypeptide which define a recognition, binding, cleavage,or other site susceptible to the activity of a protease, for example,when one or more amino acid residues encompassed by this site aresubstituted by another amino acid residue(s) or are deleted, theprotease is no longer able to cleave the TM at that site. This term alsoencompasses regions of the TM molecule which are inherently susceptibleto proteases, e.g., by being conformationally exposed and available to aprotease activity.

[0029] A “protease cleavage site” refers to the precise location atwhich a protease cleaves the TM polypeptide analog.

[0030] A “single N-terminus” and “single C-terminus” have their literalmeanings which functionally refer to the property of the composition,e.g., wherein, upon conventional sequential amino acid sequenceanalysis, each degradation cycle results in the removal of an amino acidresidue which is essentially devoid of a different amino acid residue.Thus, after several cycles, e.g., 10 cycles, of stepwise removal of theN-terminal amino acids, essentially only one amino acid is recovered ateach cycle. In particular, no more heterogeneity in sequence is detectedthan would be statistically expected from a completely pure single-chainpolypeptide according to the analytic procedure used.

[0031] “Substantially retains the biological activity of nativethrombomodulin (SEQ ID NO: 2)” and similar terms, as used herein, meansthat the thrombomodulin shares biological activities with a nativemembrane bound TM molecule. Generally, the activity in units permilligram of protein is at least about 50%, ordinarily 75%, typically85%, more typically 95%, preferably 100% and more preferably over 100%of the activity of native thrombomodulin (SEQ ID NO: 2). This biologicalactivity can be that of thrombin-mediated activation of protein C (APC),of activated partial thromboplastin clotting time (APTT), of thrombinclotting time (TCT), or of any of TM's biological, preferablytherapeutic, activities. The native standard of comparison is afull-length membrane bound version of TM, but in many cases, a solubleTM comprising the lectin/EGF/O-linked domain (TM.sub.LEO) can be used asa more convenient standard.

[0032] “Glycosylation sites” refer to amino acid residues which arerecognized by a eukaryotic cell as locations for the attachment of sugarresidues. The amino acids where sugars are attached are typically Asn(for N-linked sugars), threonine or serine (for O-linked sugars)residues. The specific site of attachment is typically signaled by asequence of amino acids, e.g., Asn—X—(Thr or Ser) for most N-linkedattachment and (Thr or Ser)—X—X—Pro for most O-linked attachment, whereX is any amino acid. The recognition sequence for glycosaminoglycans (aspecific type of sulphated sugar) is generally Ser—Gly—X—Gly, but canalso be X—Ser—Gly—X—Val. The terms N-linked and O-linked refer to thechemical group that serves as the attachment site between the sugarmoiety and the amido acid residue. N-linked sugars are attached throughan amino group; O-linked sugars are attached through an hydroxyl group.

[0033] “In vivo circulating half-life” refers to the average time ittakes an administered plasma activity in a mammal to decrease by onehalf.

[0034] A “soluble TM analog” is a TM analog which is soluble in anaqueous solution, and typically can be secreted by a cell. Forpharmacological administration, the soluble TM analog or an insolubleanalog may optionally be combined with phospholipid vesicles,detergents, or other similar compounds well known to those skilled inthe art of pharmacological formulation. The preferred TM analogs of thepresent invention are soluble in the blood stream, making the analogsuseful in various anticoagulant and other therapies. The modificationswhich make TM soluble typically do not significantly affect manyactivities relative to native thrombomodulin (SEQ ID NO: 2), e.g.,affinity for thrombin or activity in protein C activation.

[0035] “O-linked glycosylation domain” refers to the sequence of aminoacids numbered from 463 through 497 of the native thrombomodulinsequence (SEQ ID NO: 2), as depicted in Table 2 (see page 5).

[0036] “Oxidation resistant analogs” refers to analogs of thrombomodulinwhich are able to maintain a substantial amount of biological activityafter exposure to an oxidizing agent such as oxygen radicals, ChloramineT, hydrogen peroxide, or activated neutrophils.

[0037] “Solulin™ refers to a thrombomodulin analog described in U.S.Pat. No. 5,256,770, in which the following modifications to the sequenceof native thrombomodulin (SEQ ID NO: 2) have been made: removal of aminoacids 1-3, M388L, R456G, H457Q, S474A, and termination at P490.

[0038] “Pharmaceutical excipients” refers to non-toxic,medically-acceptable materials which are used to complete a medicaltherapeutic. These materials can be inert, such as water and salt, orbiologically active, such as an antibiotic or analgesic.

[0039] “Reduced ability” refers to a statistically meaningful loweringof a biological property. The property is unlimited and the measurementor quantification of the property is by standard means.

[0040] “Sugar residues” refers to hexose and pentose carbohydratesincluding glucosamines and other carbohydrate derivatives and moietieswhich are covalently linked to a protein.

[0041] “Sulfate substituents” are sulfur-containing substituents onpentose or hexose sugars.

[0042] “Thrombin-mediated conversion of fibrinogen to fibrin” refers tothe enzymatic activity by which thrombin cleaves the precursor proteinfibrinogen to make fibrin monomer, which subsequently polymerizes toform a blood clot.

[0043] “Thrombotic disease” refers to a pathogenic condition in a mammalcharacterized by the formation of one or more thrombi that are or can bedetrimental to the health of the mammal.

[0044] “SCI” as used herein refers to traumatic injuries sustained tothe spinal cord and the area around it. This includes contusion and/orcompression injuries, as well as transection injury. The model used inthe studies supporting the utility of this invention is contusion, whichmost closely approximates the types of SCI suffered by humans in motorvehicle accidents and/or sports-related injuries. All SCI ischaracterized by sudden loss of complete or partial motor function andthe extent of this loss depends on the location within the spine of theinjuries. Higher (cervical) injuries can result in total loss of motorfunction or quadraplegia and loss of respiratory control, and sometimescardiovascular collapse. Lower lesions can result in paraplegia butwithout arm involvement or respiratory dysfunction.

[0045] “Mammal” includes humans and domesticated animals, such as cats,dogs, swine, cattle, sheep, goats, horses, rabbits, and the like.

[0046] “Therapeutically effective amount” refers to that amount ofthrombomodulin analog, which, when administered to a mammal in needthereof, preferably a human, is sufficient to effect treatment, asdefined below, for neurologic damage resulting from SCI. The amount ofthrombomodulin analog which constitutes a “therapeutically effectiveamount” will vary depending on the thrombomodulin analog, the severityof SCI, and the age of the mammal to be treated, but can be determinedroutinely by one of ordinary skill in the art having regard to his ownknowledge and to this disclosure.

[0047] “Treating” or “treatment” as used herein covers amelioration ofthe neurologic damage associated with SCI in a mammal, preferably ahuman, which damage is associated with loss of motor and/or respiratoryfunction, and includes treatment which results in improved recovery ofneurologic function.

[0048] Utility of the Invention

[0049] This invention is directed to a method for treatment in mammalsof the neurologic trauma associated with spinal cord injury (SCI). Asdiscussed above, activated protein C inhibits the production of TNF-α, amolecule that has been shown to play an important role in compressiontrauma-induced SCI, and it is known that thrombomodulin, in complex withthrombin, produces activated protein C. The instant invention provides amethod for treating mammals having SCI, by administration ofthrombomodulin analogs which possess the same activity as nativethrombomodulin (SEQ ID NO: 2), but which exhibit properties which makethe analogs better therapeutic agents.

[0050] To demonstrate the utility of the thrombomodulin analogs of theinvention as therapeutic agents for treatment of the neurologic traumaassociated with SCI, thrombomodulin analogs were evaluated for theirability (1) to improve locomotor rating scale (LRS) scores, which is amethod of evaluating spinal cord function, and (2) to improve spinalcord histology, which provides a picture of spinal cord healing, in ratsfollowing SCI (see FIGS. 2-4).

[0051] As an additional indication of the utility of parenteraladministration of the thrombomodulin analog (Solulin™) in this context,it has been ascertained that intraperitoneal (i.p) administration ofSolulin™ has significant effects on plasma clotting functions.

[0052] Studies indicate that early therapeutic intervention (1-3 hrspost injury) is preferred.

[0053] Animal Models for SCI

[0054] Several experimental systems have been used to investigate thepathophysiology of SCI and to test the effects of neuroprotective agentsin the laboratory (Amar and Levy, Neurosurgery (1999), 44:1027-1040).Current experimental paradigms involve neuronal cell cultures oranatomically intact segments of spinal cord subjected to variousmechanical or ischemic insults, such as weight drop, focal orcircumferential extradural balloon compression, clip pressure,photochemical or thermal injury, distractional forces, or piston trauma.

[0055] A more preferred method which more closely approximates human SCI(Metz, et al., J. Neurotrauma

[0056] (2000), 17:1-17) is the infliction of spinal cord contusionaccording to the Multi-Center Animal Spinal Cord Injury Study (MASCIS)protocol using the controlled contusion weight drop method (Gruner, J.A, J. Neurotrauma (1992), 9:123-6; Basso, et al., J. Neurotrauma (1996),13:343-59). The resultant injury can be assessed by histologicalexamination (e.g. light or electron microscopy and special staining andtracing methods) (Gruner, J. A., Ibid.), electrophysiological outcomemeasures (e.g., evoked potentials) (Metz, et al., J. Neurotrauma (2000),17:1-17), or behavioral assessments (e.g., open field locomotion orpostural stability on an inclined plane) (Basso, et al., J. Neurotrauma(1996), 13:343-59).

[0057] Laboratory Assays for Measuring TM Activity

[0058] A number of laboratory assays for measuring TM activity areavailable. Protein C cofactor activity can be measured in the assaydescribed by Salem, et al., J. Biol. Chem. (1984), 259(19):12246-12251and Galvin, et al., J. Biol. Chem. (1987), 262(5):2199-2205. In brief,this assay consists of two steps. The first is the incubation of thetest TM analog with thrombin and protein C under defined conditions. Inthe second step, the thrombin is inactivated with hirudin orantithrombin III and heparin, and the activity of the newly activatedprotein C is determined by the use of a chromogenic substrate, wherebythe chromophore is released by the proteolytic activity of activatedprotein C. This assay is carried out with purified reagents.

[0059] Alternatively the effect of a TM analog can be measured usingplasma in clotting time assays such as the activated partialthromboplastin time (aPTT), thrombin clotting time (TCT), and/orprothrombin time (PT). The aPTT assay is dependent on both theactivating of protein C, as well as the direct inhibition of thrombin,while the TCT and PT assays are dependent only on the inhibition ofthrombin. Prolongation of the clotting time in any one of these assaysdemonstrates that the molecule can inhibit coagulation in plasma. Assayscan be run on an automatic coagulation timer according to themanufacturer's specifications; Medical Laboratory Automation Inc.distributed by American Scientific Products, McGaw Park, Ill. (See alsoSalem et al., J. Biol. Chem. (1984), 259:12246-12251, which isincorporated herein by reference).

[0060] TAFI activation can be measured as described by Wang et al. (J.Biol. Chem. (2000), 275:22942-22947), utilizing the fact that activatedTAFI is a carboxypeptidase. In this assay, extracts containing thethrombomodulin analog in question are incubated with thrombin, and themixture then incubated with purified TAFI. The amount of activated TAFIproduced is determined by the use of a chromogenic substrate, wherebythe chromophore is released by the proteolytic activity of activatedTAFI. Alternatively, TAFI activation can be assayed by a plasma clotlysis assay either in a defined system using purified proteins or in aplasma milieu (Nagashima, et al., Throm. Research (2000), 98:333-342).

[0061] The assays described above are used to identify soluble TManalogs that are able to bind thrombin and to assess the ability of thethrombin-thrombomodulin complex formed with these analogs to activateprotein C, both in purified systems and in a plasma milieu. Furtherassays can be used to evaluate other activities of native thrombomodulin(SEQ ID NO: 2) such as inhibition of thrombin catalyzed formation offibrin from fibrinogen (Jakubowski, et al., J. Biol. Chem. (1986),261(8):3876-3882), inhibition of thrombin activation of Factor V (Esmon,et al., J. Biol. Chem. (1982), 257:7944-7947), accelerated inhibition ofthrombin by antithrombin III and heparin cofactor II (Esmon, et al., J.Biol. Chem. (1983) 258:12238-12242), inhibition of thrombin activationof Factor XIII (Polgar, et al., Thromb. Haemostas. (1987), 58:140),inhibition of thrombin mediated inactivation of protein S (Thompson andSalem, J. Clin. Inv. (1986), 78(1):13-17) and inhibition of thrombinmediated platelet activation and aggregation (Esmon, et al., J. Biol.Chem. (1983), 258:12238-12242).

[0062] Modifications to Thrombomodulin

[0063] Modifications to the native thrombomodulin (SEQ ID NO: 2)molecule are useful to increase the therapeutic effectiveness of thethrombomodulin analogs of the present invention.

[0064] Particularly preferred TM analog compositions are those that haveone or more of the following characteristics:

[0065] (i) they are oxidation resistant,

[0066] (ii) they exhibit protease resistance,

[0067] (iii) they have homogeneous N- or C-termini,

[0068] (iv) they have been post-translationally modified, e.g., byglycosylation of at least some of the glycosylation sites of nativethrombomodulin (SEQ ID NO: 2),

[0069] (v) they have linear double-reciprocal thrombin bindingproperties,

[0070] (vi) they are soluble in aqueous solution in relatively lowamounts of detergents and typically lack a transmembrane sequence.

[0071] These modifications have been described in U.S. Pat. Nos.5,256,770, 5,863,760, and 5,466,668, which are each incorporated hereinby reference.

[0072] In particular, preferred modifications to the TM molecule whichrelate to these characteristics include removal of amino acids 1-13,termination at P490, and the following substitutions: M388L (foroxidation resistance), R456G and H457Q ( both for proteolysisresistance) and S474A ( blocks glycosaminoglycan addition and slowsclearance).

[0073] Most preferred is a molecule comprising all of thesemodifications, which is referred to as Solulin™.

[0074] Preparation of the TM Analogs of this Invention

[0075] Preparation of the TM analogs used in this invention is disclosedin U.S. Pat. Nos. 5,256,770, 5,863,760, and 5,466,668, which are eachincorporated herein by reference.

[0076] General Administration of Thrombomodulin Analogs

[0077] Administration of the compounds of the invention, in pure form orin an appropriate pharmaceutical composition, can be carried out via anyof the accepted modes of administration or agents for serving similarutilities. Thus, administration can be, for example, orally, nasally,parenterally, topically, transdermally, or rectally, in the form ofsolid, semi-solid, lyophilized powder, or liquid dosage forms, such asfor example, tablets, suppositories, pills, soft elastic and hardgelatin capsules, powders, solutions, suspensions, or aerosols, or thelike, preferably in unit dosage forms suitable for simple administrationof precise dosages. The compositions will include a conventionalpharmaceutical carrier or excipient and a compound of the invention asthe/an active agent, and, in addition, may include other medicinalagents, pharmaceutical agents, carriers, adjuvants, etc.

[0078] Generally, depending on the intended mode of administration, thepharmaceutically acceptable compositions will contain about 1% to about99% by weight of a compound(s) of the invention, or a pharmaceuticallyacceptable salt thereof, and 99% to 1% by weight of a suitablepharmaceutical excipient. Preferably, the composition will be about 5%to 75% by weight of a compound(s) of the invention, or apharmaceutically acceptable salt thereof, with the rest being suitablepharmaceutical excipients.

[0079] The compounds of the invention, or their pharmaceuticallyacceptable salts, may also be formulated into a suppository using, forexample, about 0.5% to about 50% active ingredient disposed in a carrierthat slowly dissolves within the body, e.g., polyoxyethylene glycols andpolyethylene glycols (PEG), e.g., PEG 1000 (96%) and PEG 4000 (4%).

[0080] The preferred route of administration is parenterally, forexample, by injection. Injection can be subcutaneous, intravenous orintramuscular. These analogs are administered in pharmaceuticallyeffective amounts and often as pharmaceutically acceptable salts, suchas acid addition salts. Such salts can include, e.g., hydrochloride,hydrobromide, phosphate, sulphate, acetate, benzoate, malate, citrate,glycine, glutamate, and aspartate, among others. See Goodman & Gilman's,The Pharmacological Basis of Therapeutics, 8^(th) ed., Pergamon Press,1985, which is incorporated herein by reference. Liquid pharmaceuticallyadministrable compositions can, for example, be prepared by dissolving,dispersing, etc., a compound(s) of the invention (about 0.5% to about20%), or a pharmaceutically acceptable salt thereof, and optionalpharmaceutical adjuvants in a carrier, such as, for example, water,saline, aqueous dextrose, glycerol, ethanol and the like, to therebyform a solution or suspension.

[0081] If desired, a pharmaceutical composition of the invention mayalso contain minor amounts of auxiliary substances such as wefting oremulsifying agents, pH buffering agents, antioxidants, and the like,such as, for example, citric acid, sorbitan monolaurate, triethanolamineoleate, butylated hydroxytoluene, etc.

[0082] Actual methods of preparing such dosage forms are known, or willbe apparent, to those skilled in sAd this art; for example, seeRemington's Pharmaceutical Sciences, 18th Ed., (Mack Publishing Company,Easton, Pa., 1990), which is incorporated herein by reference. Thecomposition to be administered will, in any event, contain atherapeutically effective amount of a compound of the invention, or apharmaceutically acceptable salt thereof, for treatment of adisease-state alleviated by the reduction of plasma levels of Lp(a) orby the inhibition of the generation of apo(a) in accordance with theteachings of this invention.

[0083] The compounds of the invention, or their pharmaceuticallyacceptable salts, are administered in a therapeutically effective amountwhich will vary depending upon a variety of factors including theactivity of the specific compound employed, the metabolic stability andlength of action of the compound, the age, body weight, general health,sex, diet, mode and time of administration, rate of excretion, drugcombination, the severity of the particular disease-states, and the hostundergoing therapy. Generally, these analogs can be administered tomammals for veterinary use, such as with domestic animals, and forclinical use in humans in a manner similar to other therapeutic agents,that is, in a physiologically acceptable carrier. In general, theadministration dosage for the TM analog will range from about at least0.0002, more usually 0.02, and less than 5000, usually less than 2000,more usually less than 500 μg/kg, usually 0.02 to 2000 μg/kg and moreusually 0.02 to 500 μg/kg of the host body weight. These dosages can beadministered by constant infusion over an extended period of time, untila desired circulating level has been attained, or preferably as a bolusinjection. Optimal dosages for a particular patient can routinely bedetermined by one of ordinary skill in the art.

[0084] Without further elaboration, it is believed that one skilled inthe art can, using the preceding description, utilize the presentinvention to its fullest extent. The following preferred specificembodiments are, therefore, to be construed as merely illustrative andnot limitative of the remainder of the disclosure in any way whatsoever.

[0085] In the foregoing and in the following examples, all temperaturesare set forth uncorrected in degrees Celsius; and, unless otherwiseindicated, all parts and percentages are by weight.

[0086] The entire disclosures of all applications, patents, andpublications, cited above and below, are hereby incorporated byreference.

[0087] While the present invention has been described with reference tothe specific embodiments thereof, it should be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made and equivalentsmay be substituted without departing from the true spirit and scope ofthe invention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

EXAMPLES Example 1

[0088] Rat Model for SCI

[0089] Animals: Adult female Sprague-Dawley rates (Charles River, N.Y.)weighing 270-325 g are housed at 12-hour light-dark cycle and fed rodentchow ad libitum and were given tap water to drink. All animalexperiments are carried out in the Animal Care Facility, under NIHGuidelines for animal studies. Only animals in good health are used. Oneweek before the surgical procedures, animals are handled on a dailybasis to adapt them for open field locomotor rating scale measurements.

[0090] Neurosurgical procedures: Animals are anesthetized with anintraperitoneal injection of ketamine (80 mg/kg) and xylazine (5 mg/kg).The wound site is prepared for contusion SCI using the NYU impactor byshaving and sterilizing the area of the incision over the dorsal lowerthoracic area according to the MASCIS protocol as described (Gruner, J.A. J. Neurotrauma (1992), 9:123-6; Basso, et al., J. Neurotrauma (1996),13: 343-59).

[0091] Prior to injury, blood pressure is monitored, arterial blood iscollected for gas measurements, and rectal temperature is recorded.

Example 2

[0092] Contusion and Post-Contusion Procedures

[0093] Contusion: Spinal cord contusion is performed using thecontrolled contusion weight-drop method with an NYU impactor, using theprotocol described above (Gruner, J. A. J. Neurotrauma (1992),9:123-126; Yong, et al., J. Neurotrauma (1998) 15:459-472).

[0094] Post-injury Procedures: During the 48 hours after injury,treatment is delivered, data collected, and blood/urine samplescollected. About a third of the rats are euthanized at 48 hours afterinjury for determination of acute lesion volume. The remainder aremaintained for from 14 to 28 days after injury to allow motor functiondeterminations (LRS/BBB) to be made.

[0095] Solulin™ Treatment: One hour after injury, a single injection of70 μg Solulin™ dissolved in 200 μl normal saline is given i.p. to agroup of three rats. Vehicle control animals, which had undergonecomplete injury as per the treatment group, received only saline. Insome experiments, a second dose of 70 μg Solulin™ dissolved in 200 μlnormal saline is given i.p. 24 h after impact. Control (sham-injured)animals underwent all surgical manipulations, including laminectomy,with the exception of weight drop injury.

[0096] aPTT measurement: Blood for plasma activated partialthromboplastin time (aPTT) levels is collected at 3, 6, 12, 24 and 72hrs after SCI. The blood is withdrawn from the tail vein using a 1 mltuberculin syringe, withdrawing approximately 1 ml of blood intopremeasured acid citrated tubes. The blood is immediately centrifuged,plasma removed, and frozen at −70° until aPTT levels can be measured(Salem et al., J. Biol. Chem. (1984), 259L12246-12251). aPTT levelmeasurements demonstrate that thrombomodulin activity is detectable forat least 24 hours post injection.

[0097] At least three separate experiments with three rats per group ofSolulin™ -treated animals and vehicle control were utilized.

[0098] Evaluation of neurologic damage: Open field locomotor ratingscale (LRS/BBB) measurements (Basso, et al., J. Neurotrauma (1996) 13:343-59; Basso et al., Exp. Neurol. (1996) 139:244-256) were performed by3 separate blinded observers over a 24 day period after SCI. Results arerecorded and entered into a software program developed for the LRS andanalyzed.

Example 3

[0099] Histological Examination of Spinal Cord Tissue

[0100] Fixed spinal cord samples are embedded and sectioned bothlongitudinally and horizontally to evaluate and measure the area of thelesion site at injury and at adjacent segments with hematoxylin andeosin (H & E), thionin and other stains. See FIG. 3. (Bethea, et al., J.Neurotrauma (1999), 16:851-63).

[0101]FIG. 4 shows an analysis of the lesion volume and indicates that astatistically significant reduction in lesion volume was found withSolulin™ treatment.

1 2 1 3466 DNA Homo sapiens CDS (151)..(1875) 1 caggggctgc gcgcagcggcaagaagtgtc tgggctggga cggacaggag aggctgtcgc 60 catcggcgtc ctgtgcccctctgctccggc acggccctgt cgcagtgccc gcgctttccc 120 cggcgcctgc acgcggcgcgcctgggtaac atg ctt ggg gtc ctg gtc ctt ggc 174 Met Leu Gly Val Leu ValLeu Gly -15 gcg ctg gcc ctg gcc ggc ctg ggg ttc ccc gca ccc gca gag ccgcag 222 Ala Leu Ala Leu Ala Gly Leu Gly Phe Pro Ala Pro Ala Glu Pro Gln-10 -5 -1 1 5 ccg ggt ggc agc cag tgc gtc gag cac gac tgc ttc gcg ctctac ccg 270 Pro Gly Gly Ser Gln Cys Val Glu His Asp Cys Phe Ala Leu TyrPro 10 15 20 ggc ccc gcg acc ttc ctc aat gcc agt cag atc tgc gac gga ctgcgg 318 Gly Pro Ala Thr Phe Leu Asn Ala Ser Gln Ile Cys Asp Gly Leu Arg25 30 35 ggc cac cta atg aca gtg cgc tcc tcg gtg gct gcc gat gtc att tcc366 Gly His Leu Met Thr Val Arg Ser Ser Val Ala Ala Asp Val Ile Ser 4045 50 ttg cta ctg aac ggc gac ggc ggc gtt ggc cgc cgg cgc ctc tgg atc414 Leu Leu Leu Asn Gly Asp Gly Gly Val Gly Arg Arg Arg Leu Trp Ile 5560 65 70 ggc ctg cag ctg cca ccc ggc tgc ggc gac ccc aag cgc ctc ggg ccc462 Gly Leu Gln Leu Pro Pro Gly Cys Gly Asp Pro Lys Arg Leu Gly Pro 7580 85 ctg cgc ggc ttc cag tgg gtt acg gga gac aac aac acc agc tat agc510 Leu Arg Gly Phe Gln Trp Val Thr Gly Asp Asn Asn Thr Ser Tyr Ser 9095 100 agg tgg gca cgg ctc gac ctc aat ggg gct ccc ctc tgc ggc ccg ttg558 Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala Pro Leu Cys Gly Pro Leu 105110 115 tgc gtc gct gtc tcc gct gct gag gcc act gtg ccc agc gag ccg atc606 Cys Val Ala Val Ser Ala Ala Glu Ala Thr Val Pro Ser Glu Pro Ile 120125 130 tgg gag gag cag cag tgc gaa gtg aag gcc gat ggc ttc ctc tgc gag654 Trp Glu Glu Gln Gln Cys Glu Val Lys Ala Asp Gly Phe Leu Cys Glu 135140 145 150 ttc cac ttc cca gcc acc tgc agg cca ctg gct gtg gag ccc ggcgcc 702 Phe His Phe Pro Ala Thr Cys Arg Pro Leu Ala Val Glu Pro Gly Ala155 160 165 gcg gct gcc gcc gtc tcg atc acc tac ggc acc ccg ttc gcg gcccgc 750 Ala Ala Ala Ala Val Ser Ile Thr Tyr Gly Thr Pro Phe Ala Ala Arg170 175 180 gga gcg gac ttc cag gcg ctg ccg gtg ggc agc tcc gcc gcg gtggct 798 Gly Ala Asp Phe Gln Ala Leu Pro Val Gly Ser Ser Ala Ala Val Ala185 190 195 ccc ctc ggc tta cag cta atg tgc acc gcg ccg ccc gga gcg gtccag 846 Pro Leu Gly Leu Gln Leu Met Cys Thr Ala Pro Pro Gly Ala Val Gln200 205 210 ggg cac tgg gcc agg gag gcg ccg ggc gct tgg gac tgc agc gtggag 894 Gly His Trp Ala Arg Glu Ala Pro Gly Ala Trp Asp Cys Ser Val Glu215 220 225 230 aac ggc ggc tgc gag cac gcg tgc aat gcg atc cct ggg gctccc cgc 942 Asn Gly Gly Cys Glu His Ala Cys Asn Ala Ile Pro Gly Ala ProArg 235 240 245 tgc cag tgc cca gcc ggc gcc gcc ctg cag gca gac ggg cgctcc tgc 990 Cys Gln Cys Pro Ala Gly Ala Ala Leu Gln Ala Asp Gly Arg SerCys 250 255 260 acc gca tcc gcg acg cag tcc tgc aac gac ctc tgc gag cacttc tgc 1038 Thr Ala Ser Ala Thr Gln Ser Cys Asn Asp Leu Cys Glu His PheCys 265 270 275 gtt ccc aac ccc gac cag ccg ggc tcc tac tcg tgc atg tgcgag acc 1086 Val Pro Asn Pro Asp Gln Pro Gly Ser Tyr Ser Cys Met Cys GluThr 280 285 290 ggc tac cgg ctg gcg gcc gac caa cac cgg tgc gag gac gtggat gac 1134 Gly Tyr Arg Leu Ala Ala Asp Gln His Arg Cys Glu Asp Val AspAsp 295 300 305 310 tgc ata ctg gag ccc agt ccg tgt ccg cag cgc tgt gtcaac aca cag 1182 Cys Ile Leu Glu Pro Ser Pro Cys Pro Gln Arg Cys Val AsnThr Gln 315 320 325 ggt ggc ttc gag tgc cac tgc tac cct aac tac gac ctggtg gac ggc 1230 Gly Gly Phe Glu Cys His Cys Tyr Pro Asn Tyr Asp Leu ValAsp Gly 330 335 340 gag tgt gtg gag ccc gtg gac ccg tgc ttc aga gcc aactgc gag tac 1278 Glu Cys Val Glu Pro Val Asp Pro Cys Phe Arg Ala Asn CysGlu Tyr 345 350 355 cag tgc cag ccc ctg aac caa act agc tac ctc tgc gtctgc gcc gag 1326 Gln Cys Gln Pro Leu Asn Gln Thr Ser Tyr Leu Cys Val CysAla Glu 360 365 370 ggc ttc gcg ccc att ccc cac gag ccg cac agg tgc cagatg ttt tgc 1374 Gly Phe Ala Pro Ile Pro His Glu Pro His Arg Cys Gln MetPhe Cys 375 380 385 390 aac cag act gcc tgt cca gcc gac tgc gac ccc aacacc cag gct agc 1422 Asn Gln Thr Ala Cys Pro Ala Asp Cys Asp Pro Asn ThrGln Ala Ser 395 400 405 tgt gag tgc cct gaa ggc tac atc ctg gac gac ggtttc atc tgc acg 1470 Cys Glu Cys Pro Glu Gly Tyr Ile Leu Asp Asp Gly PheIle Cys Thr 410 415 420 gac atc gac gag tgc gaa aac ggc ggc ttc tgc tccggg gtg tgc cac 1518 Asp Ile Asp Glu Cys Glu Asn Gly Gly Phe Cys Ser GlyVal Cys His 425 430 435 aac ctc ccc ggt acc ttc gag tgc atc tgc ggg cccgac tcg gcc ctt 1566 Asn Leu Pro Gly Thr Phe Glu Cys Ile Cys Gly Pro AspSer Ala Leu 440 445 450 gtc cgc cac att ggc acc gac tgt gac tcc ggc aaggtg gac ggt ggc 1614 Val Arg His Ile Gly Thr Asp Cys Asp Ser Gly Lys ValAsp Gly Gly 455 460 465 470 gac agc ggc tct ggc gag ccc ccg ccc agc ccgacg ccc ggc tcc acc 1662 Asp Ser Gly Ser Gly Glu Pro Pro Pro Ser Pro ThrPro Gly Ser Thr 475 480 485 ttg act cct ccg gcc gtg ggg ctc gtg cat tcgggc ttg ctc ata ggc 1710 Leu Thr Pro Pro Ala Val Gly Leu Val His Ser GlyLeu Leu Ile Gly 490 495 500 atc tcc atc gcg agc ctg tgc ctg gtg gtg gcgctt ttg gcg ctc ctc 1758 Ile Ser Ile Ala Ser Leu Cys Leu Val Val Ala LeuLeu Ala Leu Leu 505 510 515 tgc cac ctg cgc aag aag cag ggc gcc gcc agggcc aag atg gag tac 1806 Cys His Leu Arg Lys Lys Gln Gly Ala Ala Arg AlaLys Met Glu Tyr 520 525 530 aag tgc gcg gcc cct tcc aag gag gta gtg ctgcag cac gtg cgg acc 1854 Lys Cys Ala Ala Pro Ser Lys Glu Val Val Leu GlnHis Val Arg Thr 535 540 545 550 gag cgg acg ccg cag aga ctc tgagcggcctccgtccagga gcctggctcc 1905 Glu Arg Thr Pro Gln Arg Leu 555 gtccaggagcctgtgcctcc tcacccccag ctttgctacc aaagcacctt agctggcatt 1965 acagctggagaagaccctcc ccgcaccccc caagctgttt tcttctattc catggctaac 2025 tggcgagggggtgattagag ggaggagaat gagcctcggc ctcttccgtg acgtcactgg 2085 accactgggcaatgatggca attttgtaac gaagacacag actgcgattt gtcccaggtc 2145 ctcactaccgggcgcaggag ggtgagcgtt attggtcggc agccttctgg gcagaccttg 2205 acctcgtgggctagggatga ctaaaatatt tatttttttt aagtatttag gtttttgttt 2265 gtttcctttgttcttacctg tatgtctcca gtatccactt tgcacagctc tccggtctct 2325 ctctctctacaaactcccac ttgtcatgtg acaggtaaac tatcttggtg aatttttttt 2385 tcctagccctctcacattta tgaagcaagc cccacttatt ccccattctt cctagttttc 2445 tcctcccaggaactgggcca actcacctga gtcaccctac ctgtgcctga ccctacttct 2505 tttgctcttagctgtctgct cagacagaac ccctacatga aacagaaaca aaaacactaa 2565 aaataaaaatggccatttgc tttttcacca gatttgctaa tttatcctga aatttcagat 2625 tcccagagcaaaataatttt aaacaaaggt tgagatgtaa aaggtattaa attgatgttg 2685 ctggactgtcatagaaatta cacccaaaga ggtatttatc tttactttta aacagtgagc 2745 ctgaattttgttgctgtttt gatttgtact gaaaaatggt aattgttgct aatcttctta 2805 tgcaatttccttttttgtta ttattactta tttttgacag tgttgaaaat gttcagaagg 2865 ttgctctagattgcgagaag agacaaacac ctcccaggag acagttcaag aaagcttcaa 2925 actgcatgattcatgccaat tagcaattga ctgtcactgt tccttgtcac tggtagacca 2985 aaataaaaccgactctactg gtcttgtgga attgggagct tgggaatgga tcctggagga 3045 tgcccaattagggcctagcc ttaatcaggt cctcagagaa tttctaccat ttcagagagg 3105 ccttttggaatgtggcccct gaacaagaat tggaagctgc cctgcccatg ggagctggtt 3165 agaaatgcagaatcctaggc tccaccccat ccagttcatg agaatctata tttaacaaga 3225 tctgcagggggtgtgtctgc tcagtaattt gaggacaacc attccagact gcttccaatt 3285 ttctggaatacatgaaatat agatcagtta taagtagcag gccaagtcag gcccttattt 3345 tcaagaaactgaggaatttt ctttgtgtag ctttgctctt tggtagaaaa ggctaggtac 3405 acagctctagacactgccac acagggtctg caaggtcttt ggttcagcta agccggaatt 3465 c 3466 2 575PRT Homo sapiens 2 Met Leu Gly Val Leu Val Leu Gly Ala Leu Ala Leu AlaGly Leu Gly -15 -10 -5 Phe Pro Ala Pro Ala Glu Pro Gln Pro Gly Gly SerGln Cys Val Glu -1 1 5 10 His Asp Cys Phe Ala Leu Tyr Pro Gly Pro AlaThr Phe Leu Asn Ala 15 20 25 30 Ser Gln Ile Cys Asp Gly Leu Arg Gly HisLeu Met Thr Val Arg Ser 35 40 45 Ser Val Ala Ala Asp Val Ile Ser Leu LeuLeu Asn Gly Asp Gly Gly 50 55 60 Val Gly Arg Arg Arg Leu Trp Ile Gly LeuGln Leu Pro Pro Gly Cys 65 70 75 Gly Asp Pro Lys Arg Leu Gly Pro Leu ArgGly Phe Gln Trp Val Thr 80 85 90 Gly Asp Asn Asn Thr Ser Tyr Ser Arg TrpAla Arg Leu Asp Leu Asn 95 100 105 110 Gly Ala Pro Leu Cys Gly Pro LeuCys Val Ala Val Ser Ala Ala Glu 115 120 125 Ala Thr Val Pro Ser Glu ProIle Trp Glu Glu Gln Gln Cys Glu Val 130 135 140 Lys Ala Asp Gly Phe LeuCys Glu Phe His Phe Pro Ala Thr Cys Arg 145 150 155 Pro Leu Ala Val GluPro Gly Ala Ala Ala Ala Ala Val Ser Ile Thr 160 165 170 Tyr Gly Thr ProPhe Ala Ala Arg Gly Ala Asp Phe Gln Ala Leu Pro 175 180 185 190 Val GlySer Ser Ala Ala Val Ala Pro Leu Gly Leu Gln Leu Met Cys 195 200 205 ThrAla Pro Pro Gly Ala Val Gln Gly His Trp Ala Arg Glu Ala Pro 210 215 220Gly Ala Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys 225 230235 Asn Ala Ile Pro Gly Ala Pro Arg Cys Gln Cys Pro Ala Gly Ala Ala 240245 250 Leu Gln Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gln Ser Cys255 260 265 270 Asn Asp Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp GlnPro Gly 275 280 285 Ser Tyr Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu AlaAla Asp Gln 290 295 300 His Arg Cys Glu Asp Val Asp Asp Cys Ile Leu GluPro Ser Pro Cys 305 310 315 Pro Gln Arg Cys Val Asn Thr Gln Gly Gly PheGlu Cys His Cys Tyr 320 325 330 Pro Asn Tyr Asp Leu Val Asp Gly Glu CysVal Glu Pro Val Asp Pro 335 340 345 350 Cys Phe Arg Ala Asn Cys Glu TyrGln Cys Gln Pro Leu Asn Gln Thr 355 360 365 Ser Tyr Leu Cys Val Cys AlaGlu Gly Phe Ala Pro Ile Pro His Glu 370 375 380 Pro His Arg Cys Gln MetPhe Cys Asn Gln Thr Ala Cys Pro Ala Asp 385 390 395 Cys Asp Pro Asn ThrGln Ala Ser Cys Glu Cys Pro Glu Gly Tyr Ile 400 405 410 Leu Asp Asp GlyPhe Ile Cys Thr Asp Ile Asp Glu Cys Glu Asn Gly 415 420 425 430 Gly PheCys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys 435 440 445 IleCys Gly Pro Asp Ser Ala Leu Val Arg His Ile Gly Thr Asp Cys 450 455 460Asp Ser Gly Lys Val Asp Gly Gly Asp Ser Gly Ser Gly Glu Pro Pro 465 470475 Pro Ser Pro Thr Pro Gly Ser Thr Leu Thr Pro Pro Ala Val Gly Leu 480485 490 Val His Ser Gly Leu Leu Ile Gly Ile Ser Ile Ala Ser Leu Cys Leu495 500 505 510 Val Val Ala Leu Leu Ala Leu Leu Cys His Leu Arg Lys LysGln Gly 515 520 525 Ala Ala Arg Ala Lys Met Glu Tyr Lys Cys Ala Ala ProSer Lys Glu 530 535 540 Val Val Leu Gln His Val Arg Thr Glu Arg Thr ProGln Arg Leu 545 550 555

What is claimed is:
 1. A method for treating the neurologic damageresulting from spinal cord injury in a mammal, which method comprisesadministering to said mammal in need thereof a therapeutically effectiveamount of a soluble, recombinant thrombomodulin analog which isresistant to oxidation and wherein the methionine at position 388 hasbeen replaced with a leucine, wherein the analog is numbered inaccordance with native thrombomodulin (SEQ ID NO: 2).
 2. The method ofclaim 1, wherein the thrombomodulin analog is modified in the sugarresidues of the O-linked glycosylation domain of native thrombomodulin(SEQ ID NO: 2).
 3. The method of claim 2, wherein the thrombomodulinanalog is modified such that the O-linked glycosylation domain has nochondroitin sulfate.
 4. The method of claim 1, wherein the analog hasbeen rendered resistant to protease cleavage.
 5. The method of claim 1,wherein the thrombomodulin analog (Solulin™) has the amino acid sequenceof native thrombomodulin (SEQ ID NO: 2) modified at the followingpositions: removal of amino acids 1-3 M388L R456G H457Q S474A, andterminating at P490.
 6. The method of claim 5 wherein the mammal in needthereof is a human.
 7. A pharmaceutical composition useful in treatingneurologic damage resulting from spinal cord injury in a mammal, whichpharmaceutical composition comprises a pharmaceutical excipient and atherapeutically effective amount of a soluble, recombinantthrombomodulin analog which is resistant to oxidation and wherein themethionine at position 388 has been replaced with a leucine, wherein theanalog is numbered in accordance with native thrombomodulin (SEQ ID NO:2).
 8. The pharmaceutical composition of claim 7, wherein thethrombomodulin analog is modified in the sugar residues of the O-linkedglycosylation domain of native thrombomodulin (SEQ ID NO: 2).
 9. Thepharmaceutical composition of claim 8, wherein the thrombomodulin analogis modified such that the O-linked glycosylation domain has nochondroitin sulfate.
 10. The pharmaceutical composition of claim 7,wherein the analog has been rendered resistant to protease cleavage. 11.The pharmaceutical composition of claim 7, wherein the thrombomodulinanalog (Solulin™) has the amino acid sequence of native thrombomodulin(SEQ ID NO: 2) modified at the following positions: removal of aminoacids 1-3 M388L R456G H457Q S474A, and terminating at P490.